One step 64Cu-BaBaSar-RGD2 production method

ABSTRACT

A method of preparing a  64 Cu-BaBaSar-RGD 2  solution is provided. The method includes lyophilizing a solution of BaBaSar-RGD 2  and adding a  64 Cu solution to the lyophilized BaBaSar-RGD 2 .

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. § 119(e) ofU.S. Ser. No. 62/593,723, filed Dec. 1, 2017, the entire contents ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates in general to the preparation of imagingprobes.

BACKGROUND OF THE INVENTION

Tumor-induced angiogenesis plays a critical role in tumor progressionand metastasis. Without new vasculature and blood circulation, tumorstops growing at the size of 1-2 mm3 and may become necrotic or evenapoptotic since diffusion is already insufficient to supply the tissuewith oxygen and nutrients (1, 2). Substantial efforts have been made todevelop therapeutic strategies that interrupt the angiogenic process tostop the tumor growth (3). Integrin αvβ3 is a vital component for theangiogenic process by mediating endothelial cell (EC) migration andsurvival during angiogenesis (4). For neovasculature formation, ECs needto migrate into an avascular region and to extensively remodel theextracellular matrix (ECM). In this process, integrins αvβ3, animmunoglobulin superfamily molecule has proved to be one of the mostimportant cell adhesion receptors for various ECM proteins. While innormal tissues, expression of integrin αvβ3 is much lower, makingintegrin αvβ3 an ideal target for diagnosis and therapy in cancer study.A protocol to non-invasively quantify its expression levels will providea method to document integrin levels, which can support theanti-integrin αvβ3 treatment for the patients, and effectively monitortreatment progress for the integrin αvβ3-positive patients. Non-invasivedetection and quantification of integrin αvβ3 is also leading to thediagnosis of many types of cancer at their earliest stages (5).

Peptides containing Arg-Glu-Asp (RGD) amino acid sequence have a highbinding affinity and selectivity for integrin αvβ3 (6). In the last twodecades, a number of peptides containing RGD sequences have beendeveloped to target tumors overexpressing α_(v)β₃ receptors (7). RGDpeptides have been modified and radiolabeled for positron emissiontomography (PET) probe development. ¹⁸F-galacto-RGD is the first RGDprobe tested in human subjects for detecting α_(v)β₃ expression. Withconjugation of a sugar moiety for reducing the liver uptake,¹⁸F-galacto-RGD is still specifically binding to integrin α_(v)β₃, showsa more desirable biodistribution in humans, and provides a bettervisualization of α_(v)β₃ expression in tumors with high contrast (8).However, a major disadvantage for ¹⁸F-galacto-RGD is the long andsophisticated preparation, including multiple synthetic steps thatcomplicate routine production (9). Due to the importance of RGDpeptides, continued efforts have been made to achieve desirable PETprobes for easy production, optimal pharmacokinetics, and higher tumoruptake, such as ¹⁸F-AH111585 (10-12), ¹⁸F-alfatide (13,14), ¹⁸F-RGD-K5(15,16), ¹⁸F-FPPRGD₂ (17,18), ¹⁸F-fluciclatide (12,19), and⁶⁸Ga-NOTA-PRGD₂ (20).

⁶⁴Cu (T_(1/2)=12.7 h; β⁺ 0.653 MeV [17.8%]) has been widely used forradiolabeling proteins, antibodies and peptides for PET probedevelopment. The low β⁺ energy of ⁶⁴Cu gives a resolution down to 1 mmin PET images and is important to achieve lower radiation doses for thepatients (21). Cage-like hexaazamacrobicyclic sarcophagine chelatorcompletely encapsulates the coordinated Cu²⁺ ions. Their complexesexhibit enhanced thermodynamic and kinetic stability to copper-bindingproteins in vivo (22). Starting from hexaazamacrobicyclic sarcophagine,a BaBaSar chelator for conjugation with RGD peptide (BaBaSar-RGD₂) wasdeveloped. The ⁶⁴Cu labeling chemistry for BaBaSar-RGD₂ was achieved atroom temperature to give a quantitative yield. The resulting⁶⁴Cu-BaBaSar-RGD₂ probe shows great stability both in vitro and in vivo,providing high tumor uptake and low normal organ uptake in U87MGglioblastoma tumor bearing mice (23). Due to the wide application of RGDpeptide in diagnostic and therapeutic applications, there is a need forPET radiotracer for integrin imaging that can be made easily. Such a PETradiotracer would be of great interest to both radiochemists andphysicians.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a method of preparinga ⁶⁴Cu-BaBaSar-RGD₂ solution. The method includes lyophilizing asolution of BaBaSar-RGD₂ and adding a ⁶⁴Cu solution to the lyophilizedBaBaSar-RGD₂.

In one embodiment, the ⁶⁴Cu solution includes ⁶⁴CuCl₂.

In another embodiment, the ⁶⁴Cu solution includes buffer salts.

In another embodiment, the solution of BaBaSar-RGD₂ includes buffersalts.

In another embodiment, the buffer salts include sodium acetate buffer.

Another aspect of the present invention is directed to a methodpreparing a ⁶⁴Cu-BaBaSar-RGD₂ solution. The method includes lyophilizinga ⁶⁴Cu-BaBaSar-RGD₂ solution and reconstituting the ⁶⁴Cu-BaBaSar-RGD₂solution with an aqueous solution.

In one embodiment, the ⁶⁴Cu-BaBaSar-RGD₂ solution includes buffer salts.

In another embodiment, the buffer salts include sodium acetate buffer.

Another aspect of the present invention is to provide a kit forpreparing a positron emission tomography (PET) probe. The kit includes alyophilized powder of BaBaSar-RGD₂ and instructions on how toreconstitute the lyophilized powder with a ⁶⁴Cu solution.

In some embodiments, the ⁶⁴Cu solution comprises a ⁶⁴Cu²⁺ salt dissolvedor dispersed in a suitable liquid medium. The ⁶⁴Cu solution comprises a⁶⁴Cu halide salt, wherein the halide is selected from a group thatincludes fluorine, chlorine, bromine and iodine.

In some embodiments, the ⁶⁴Cu solution can include one or more buffersalts.

In some embodiments, the solution of BaBaSar-RGD₂ can include one ormore buffer salts.

Other aspects and advantages of the invention will be apparent from thefollowing description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Kit production process of ⁶⁴Cu-BaBaSar-RGD₂.

FIG. 2: Structure of RGD peptide and the synthesis route for⁶⁴Cu-BaBaSar-RGD₂.

FIG. 3: Analytical radio trace HPLC chromatogram for the purity of the⁶⁴Cu-BaBaSar-RGD₂.

FIG. 4: Decay-corrected anterior maximum-intensity projections of PET/CTat 1, 5, 10, 20, 40, 60, 120, and 180 min after injection of⁶⁴Cu-BaBaSar-RGD₂ in macaque monkey.

FIG. 5: Structures of AnAnSar, BaAnSar, BaMalSar, and MalMalSar.

FIG. 6: Additional peptides to be used in present invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated herein, all terms used herein have themeanings that the terms would have to those skilled in the art of thepresent invention. Practitioners are particularly directed to currenttextbooks for definitions and terms of the art. It is to be understood,however, that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary.

To promote the clinical application of ⁶⁴Cu-BaBaSar-RGD₂ in humans, thepresent invention provides a straightforward, one-step synthesis of⁶⁴Cu-BaBaSar-RGD₂ radiopharmaceutical using a preloaded cold kit.

The present invention provides a one-step production ofradiopharmaceutical ⁶⁴Cu-BaBaSar-RGD₂ with a kit preloaded with all theprecursors. FIG. 1 discloses the process of production. Furthermore,this method is not limited to the production of ⁶⁴Cu-BaBaSar-RGD₂. Whenbiological ligands other than RGD peptides are conjugated with theBaBaSar chelator, the same kit method associated with BaBaSar chelatorcould be used too. For example, other peptides can be used in place ofRGD. In addition, other chelators, preferably sarcophagine basedchelators, such as AnAnSar, BaAnSar, BaMalSar, and MalMalSar can be usedin place of BaBaSar. Therefore, this kit method provides a universalmethod for ⁶⁴Cu radiopharmaceutical production.

General Method

BaBaSar-RGD₂ was synthesized as previously reported (23). All commercialchemicals were of analytic grade and used without further purification.⁶⁴Cu in hydrochloric acid was obtained from Washington University (St.Louis, Mo.) or produced in the Molecular Imaging Center CyclotronFacility. Analytic reversed-phase high-performance liquid chromatography(RP-HPLC) using a Phenomenex Luna column (5μ, C₁₈, 250×4.6 mm) wereperformed on a Dionex U3000 chromatography system with a diode arraysdetector and radioactivity flow-count (Eckert & Ziegler, Valencia,Calif.). The recorded data were processed using Chromeleon version 7.20software. The flow rate of analytical HPLC was 1.0 mL/min. The mobilephase starts from 95% solvent A (0.1% trifluoroacetic acid [TFA] inwater) and 5% solvent B (0.1% TFA in acetonitrile [MeCN]). From 2 to 32min, the mobile phase ramped to 35% solvent A and 65% solvent B. Theultraviolet (UV) detector of HPLC was set at 254 nm. The endotoxinanalysis was performed on a portable Endosafe®-PTS™ system consisting ofLAL reagent and endotoxin controls applied to a single use, polystyrenecartridge.

Radiopharmaceutical Preparation

Preparation of ⁶⁴Cu-BaBaSar-RGD₂ Production Kit

The 18.2 MΩ·cm water from in-house GenPure™ station was treated withchelex 100 resin 48 hours before use. All the solution hereafter wasprepared with this treated water. The lyophilized BaBaSar-RGD₂ (1.0 mg)was dissolved in 1.0 mL sodium acetate buffer (NaOAc, 0.1 M, pH 5.5).The pH of BaBaSar-RGD₂ solution was adjusted to pH 5.5 using 0.1 Msodium hydroxide (NaOH). Then, the BaBaSar-RGD₂ solution was equallyaliquoted to 20 Eppendorf vials (1.5 mL). The filled vials were frozenusing dry ice and then transferred to the bottles of the Labconco FreezeDry System (pressure <100 mTorr). After the solvent was removed, thevials containing BaBaSar-RGD₂ powder were then sealed and stored at −18°C. for ⁶⁴Cu labeling.

⁶⁴Cu-Labeling Chemistry

64CuCl2 (5-30 mCi) purchased from Washington University at St. Louis wasreconstituted using 200-300 μL NaOAc buffer (0.1 M, pH 5.5) and added toa vial prepared in above section. The vial was gently shaking at roomtemperature for 5 min. After the reaction was quenched with 5.0 mLsaline, the activity passed through a 0.22 μm sterile filter (PallCorp.) into a 10 mL Allergy vial for quality control test andanimal/human injection.

Kit Preparation

A cold kit can contain 50 μg BaBaSar-RGD₂ ligand to which ⁶⁴CuCl₂ is tobe complexed, and buffer salts to adjust the pH suitable for thelabelling conditions. The kits are prepared in a lyophilized form andhave a long shelf life of over 3 months at room temperature. When thecold kits are stored in a refrigerator at 2-8° C., the shelf life isover a year.

Radiochemistry

The labeling chemistry for ⁶⁴Cu-BaBaSar-RGD₂ is disclosed in FIG. 2. The⁶⁴Cu-labeling yield for ⁶⁴Cu-BaBaSar-RGD₂ was >99% based on HPLCanalysis (FIG. 3). However, after passing through 0.22 μm Pall filter toremove pyrogen, approximately 15-20% ⁶⁴Cu-BaBaSar-RGD₂ was trapped ontothe filter and the overall recovered yield for ⁶⁴Cu-BaBaSar-RGD₂ isabout 80% calculated from the loaded ⁶⁴Cu. The radiochemical purity of⁶⁴Cu-BaBaSar-RGD₂ was >99% based on radiotrace analytical HPLC (FIG. 3).The retention times for free ⁶⁴CuCl₂ and ⁶⁴Cu-BaBaSar-RGD₂ on HPLC were2.5 and 13.9 min, respectively. The reaction crude without purificationsdid not show free ⁶⁴Cu in HPLC chromatograms. Therefore, no furtherpurification is needed for the final product.

Quality Control

All the quality control results met the pre-specified limits for 3validation runs. These included half-life, appearance, pH value,identity, endotoxin amount, etc. (Table 1). The specific activitydetermined by HPLC analysis was between 389.2 and 605.4 mCi/μmol(average±SD, 473.0±116.2 mCi/μmol). Therefore, a human dose (<25 mCi) of⁶⁴Cu-BaBaSar-RGD₂ contained less than 125 μg of RGD peptide.

TABLE 1 Quality control data from 3 synthesis runs. QC Test ReleaseCriteria Run 1 Run 2 Run 3 Product (mCi) none 5.5 6.2 4.5 VisualInspection Clear, colorless Yes Yes Yes Radiochemical Identity RRT =0.9-1.1 1.0 1.0 1.0 Radiochemical Purity >90% 99% 100% 99% SpecificActivity >100  15.7 14.4 22.4 (mCi/μmol) Dose pH 4.5-7.5 5.5 6.0 6.0Sterile Filter >45 64 64 62 Integrity Test (psi) Radionuclidic Identity12.6-12.8 h 12.7 12.7 12.7 (t_(1/2)) Endotoxin Analysis  ≤17.5 <5 <5 <5(EU/mL)Absorbed Dose Estimates from Macaque Imaging

The injection of 13.1-19.7 MBq/kg of ⁶⁴Cu-BaBaSar-RGD₂ in macaque monkeyproduced no observable effects on vital signs (blood pressure, pulse,and electrocardiogram) during and 24-h after PET scan. The PET images at1, 5, 10, 20, 40, 60, 120, and 180 min after injection are disclosed inFIG. 4. At 1 min, rapid uptake of ⁶⁴Cu-BaBaSar-RGD₂ was observed in theheart, and liver. The bladder content was visualized at 10 min afterinjection and more and more activity was accumulated in urine bladdercontent. The bladder did not void because the macaque monkey was underanesthesia. Gallbladder uptake was not observed during the whole scan.Rapid clearance of activity in the liver was observed in the images attime points after 1 min. The urinary bladder had the highest uptake,with 51.37%±8.73% of injected activity at 1 h post injection. Themaximum uptake for the liver, and kidneys were 37.40±6.63% ID (9 min)and 26.79±4.35% ID (0.5 min) respectively. At 3 h of post injection,8.62%±1.41% of injected activity was found in the gallbladder, smallintestine, and upper and lower portions of the large intestine.

The mean organ doses for the male human phantom were calculated withOlinda/EXM using ⁶⁴Cu-BaBaSar-RGD₂ biodistribution in monkey (Table 2).The kidneys had the highest radiation-absorbed doses (108.43 μGy/MBq),followed by the bladder wall (87.07 μGy/MBq). The mean effective dose of⁶⁴Cu-BaBaSar-RGD₂ was 15.30±2.21 μSv/MBq. When 925-MBq of⁶⁴Cu-BaBaSar-RGD₂ is administrated into human subject, the effectivedose for the non-voiding model is estimated to be 14.2 mSv, which iscomparable to the estimated 6.23 mSv dose in a whole-body PET scan with2-deoxy-2-[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) (24). The estimated doses forthe female human were higher by 18% because body and organ sizes ofwomen are smaller than those men (data not shown).

Venous blood samples were withdrawn from monkey during the PET scan.Based on the decay corrected activity per unit of blood sample, it wasfound that ⁶⁴Cu-BaBaSar-RGD₂ was cleared rapidly from the blood. By 3 hafter injection, 2.88±0.88% ID remained (range, 2.07-3.82% ID). At 22 hafter injection, the activity in the blood decreased to 0.79±0.52% ID.Based on the percentage of injected dose in blood sample, the half lifeof ⁶⁴Cu-BaBaSar-RGD₂ in blood pool was calculated as 12.1±4.0 min (n=3).

TABLE 2 Estimated Human Absorbed Doses of ⁶⁴Cu-BaBaSar-RGD₂ to NormalOrgans Using Biodistribution Data from Macaque monkey. Organs Mean ± SD(μGy/MBq) Adrenals 3.34 ± 0.52 Brain 1.27 ± 0.22 Breasts 1.34 ± 0.23Gall bladder Wall 3.07 ± 0.49 LLI Wall 2.86 ± 0.44 Small Intestine 4.53± 0.68 Stomach Wall 2.11 ± 0.34 ULI Wall 2.47 ± 0.39 Heart Wall 4.39 ±0.62 Kidneys 108.43 ± 16.41  Liver 7.54 ± 1.15 Lungs 1.67 ± 0.28 Muscle1.88 ± 0.31 Ovaries 2.88 ± 0.44 Pancreas 2.86 ± 0.45 Red Marrow 9.29 ±1.02 Osteogenic Cells 7.01 ± 0.91 Skin 1.38 ± 0.24 Spleen 6.78 ± 0.88Testes 2.03 ± 0.33 Thymus 1.56 ± 0.26 Thyroid 1.39 ± 0.24 UrinaryBladder Wall 87.07 ± 12.38 Uterus 4.16 ± 0.63 Total Body 2.76 ± 0.42Effective Dose* 15.30 ± 2.21  *In unit of μSv/MBq

Integrin αvβ3-targeted radiopharmaceutical ⁶⁴Cu-BaBaSar-RGD₂ has beensuccessfully synthesized with the one-step kit method. Thestraightforward method greatly simplifies the production process andbenefits the clinical application of ⁶⁴Cu-BaBaSar-RGD₂. Human radiationdosimetry of ⁶⁴Cu-BaBaSar-RGD₂ was estimated after intravenousadministration in macaque monkey, by PET imaging and OLINDA/EXMcalculations. The critical organs were kidneys and urinary bladder wall.The mean effective dose, determined with the male adult model, was15.30±2.21 Sv/MBq. This PET probe demonstrates an acceptable radiationdose comparable to other reported RGD-derived radiopharmaceuticals.These demonstrate great promise of ⁶⁴Cu-BaBaSar-RGD₂ as an integrinmarker, with a desirable biodistribution and safety characteristics inmonkey. Therefore, ⁶⁴Cu-BaBaSar-RGD₂ can safely be used in human scanfor further evaluation of its performance as an integrin-targetingprobe.

REFERENCES

All references cited herein, including those below and including but notlimited to all patents, patent applications, and non-patent literaturereferenced below or in other portions of the specification, are herebyincorporated by reference herein in their entirety.

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What is claimed is:
 1. A method of preparing a ⁶⁴Cu-BaBaSar-RGD₂solution comprising: lyophilizing a solution of BaBaSar-RGD₂ comprisinga buffer salt to obtain a powder of BaBaSar-RGD₂ and the buffer salt;and adding a ⁶⁴Cu solution comprising ⁶⁴CuCl₂ to the powder ofBaBaSar-RGD₂ and the buffer salt.
 2. The method of claim 1, wherein thebuffer salt is sodium acetate buffer.